Medicine response assay in respiratory disease

ABSTRACT

Correlations between polymorphisms in the 5-lipoxygenase gene, or polymorphisms in the leukotriene C4 synthase gene, and a subject&#39;s phenotypic response to treatment with a leukotriene receptor antagonist for respiratory disease are described. Methods of screening subjects to aid in treatment, and methods of screening therapeutic compounds, are presented.

This application is filed pursuant to 35 U.S.C. § 371 as a United StatesNational Phase Application of International Application No.PCT/US01/12534 filed Apr. 17, 2001, which claims priority from U.S.Provisional Application No. 60/197,913 filed Apr. 17, 2000, and U.S.Provisional Application No. 60/236,608 filed Sep. 29, 2000.

FIELD OF THE INVENTION

The present studies relate to polymorphisms in genes that play a role inthe biosynthesis of sulfidopeptide leukotrienes, and phenotypes that areassociated or correlated therewith. More particularly, the presentstudies relate to the correlation of such polymorphisms to the responseof subjects with respiratory disorders (such as asthma) topharmaceutical treatment. The present studies further relate to methodsof screening compounds for pharmaceutical activity. The present studiesalso relate to methods of genotyping subjects for predictive purposes.

BACKGROUND OF THE INVENTION

Asthma is an extremely common disorder accounting for 1 to 3% of alloffice visits, 500,000 hospital admissions per year and more pediatrichospital admissions than any other single illness. Asthma is no longersimply viewed as reversible airway obstruction or irritable airways.Rather, it is viewed primarily as an inflammatory illness that hasbronchial hyperreactivity and bronchospasm as a result. The asthmaticinflammation that underlies the disease can be addressed by therapy withanti-inflammatory agents such as glucocorticoids or by those agents thattarget the role of the leukotrienes in the inflammation process.

The leukotrienes are a family of eicosatetraenoic acids derived fromarachidonic acid which exhibit a wide range of pharmacological andphysiological activities including bronchoconstriction andproinflammatory activity. Arachidonic acid is cleaved from the cellmembrane, and acted upon by a cascade of enzymes localized to theperinuclear membrane including 5-lipoxygenase (ALOX5) which forms anunstable epoxide LTC4, followed by the addition of glutathione by LTC4Synthase (LTC4S) forming the intercellular LTC4. LTC4 is transportedextracellularly, where an amino acid is sequentially cleaved to formcystinyl leukotrienes LTD4 and LTE4. The cystinyl leukotrienes LTD4 andLTE4 act through the CYS-LT1 receptor. Two classes of drugs exist tomodulate this pathway, ALOX5 inhibitors and CYSLT1 receptor antagonists.

A polymorphism in the promoter region of the ALOX5 gene has beendemonstrated to affect the transcription of this gene. In vitro, humancells containing other than 5 random repeats of the Sp-1 binding motif(GGGCGG) have diminished activity. It follows that patients with asthmathat have a variant genotype would be less responsive to therapeuticintervention with agents modifying this pathway. Prior clinical trialswith the ALOX5 inhibitor compound ABT-761 have determined that efficacyof the compound is affected by genotype (Drazen, J. M. et alPharmacogenetic association between ALOX5 promoter genotype and theresponse to anti-asthma treatment. Nature Genetics 22;168-170, 1999).Patients that were either homozygous for the wild type allele (5 tandemrepeats of the Sp-1 motif) or heterozygous (5/any other number of tandemrepeats) had similar efficacy. However, the patients that werehomozygous for no wild type alleles (variants) had a greatly reducedresponse to the compound. See FIG. 1 (which graphs data provided inDrazen et al. (1999) at page 168, column 2, last paragraph, continuingto page 169). In FIG. 1, homozygous for the wild type allele is denoted5,5; heterozygous the wild and variant alleles is denoted 5,X; andhomozygous for variant alleles is denoted X,X.

There is a need for a method of determining whether a given asthmapatient would or would not be a good candidate for treatment with aleukotriene pathway modulator. It is therefore a goal of the presentinvention to provide an association between optimum clinical outcome ofpharmacologic therapy and the genotype of the individual population. Itis another goal of the present invention to be able to stratify patientpopulations into those subsets that will respond to a givenpharmacologic therapy more or less well relative to other pharmacologictherapies. Another goal of the present invention is to be able topredict a patient's response to a given pharmacologic therapy on thebasis of that patient's genotype. An additional goal of the presentinvention is to provide for a commercial method of predicting patientresponses to pharmacologic therapies. Yet another goal of the presentinvention is to provide a method of screening candidate drug compoundsfor future suitable administration to a patient or to a patientpopulation, based upon the genotype of the patient or the population.This entails a method of screening candidate drug compounds forvariations in a measurable phenotypic effect among geneticsubpopulations of subjects with asthma.

SUMMARY OF THE INVENTION

The present inventors have determined that in subjects with respiratorydisorders that are potentially treatable with leukotriene receptorantagonists, polymorphisms in the 5-lipoxygenase (ALOX5) gene and/or theleukotriene C₄ synthase gene (LTC4S) are correlated with the response ofthe subjects to pharmacologic therapy with a leukotriene receptorantagonist. More particularly, they have found that a transversionpolymorphism in the promoter region of the LTC4S gene is a predictor forthe response of patients with asthma to treatment with a CysLT1leukotriene receptor antagonist, and they have found that a polymorphicvariation in the number of tandem repeats of a Sp-1 binding motif in thepromoter region of the ALOX5 gene, as well as other transversionpolymorphisms, likewise are predictors for the response of patients withasthma to treatment with a CysLT1 leukotriene receptor antagonist; andfurthermore, they have identified a genetic subset of asthma patientswho display a lower incidence of relief of asthma symptoms when treatedwith a leukotriene receptor antagonist as compared to alternativepharmacologic therapies.

A first aspect of the present invention is a method of screening apatient population to identify those subjects with an decreasedlikelihood of responding favorably to treatment with a leukotrieneantagonist for a respiratory disease such as asthma. The subjects mayhave been previously diagnosed with the respiratory disease, or thescreening may be used in conjunction with diagnostic efforts.

A further aspect of the present invention is a method of screening asubject with a respiratory disease that is treatable with leukotrienereceptor antagonists (such as asthma), as an aid in predicting thesubject's response to treatment with a leukotriene receptor antagonist.The method comprises obtaining a sample of the subject's DNA anddetermining the genotype of the subject at a polymorphic allelic site ineither one or both of the ALOX5 gene or the LTC4S gene, where differentgenotypes at that site have been associated with different incidences ofa phenotypic response to treatment with a leukotriene receptorantagonist.

A further aspect of the present invention is a method of screening asubject suffering from asthma that is treatable with a leukotrienereceptor antagonist ligand, as an aid in predicting the subject'sresponse to treatment with that leukotriene receptor antagonist. Themethod comprises obtaining a sample of the subject's DNA and determiningthe genotype of the subject at a polymorphic allelic site in either orboth of the ALOX5 gene and the LTC4S gene, where different genotypes atthat site have been associated with different incidences of a phenotypicresponse to treatment with a leukotriene receptor antagonist. Thegenotype that is detected in the sample indicates that the subject islikely to have the phenotypic response associated with that genotype.

A further aspect of the present invention is a method of screening aligand for variations in measurable phenotypic effects among geneticsubpopulations of subjects with a respiratory disorder. The methodcomprises administering the candidate ligand to a population of subjectssuffering from the respiratory disorder, and obtaining DNA samples fromeach of the subjects. The DNA samples are genotyped for a polymorphicallele of the ALOX5 gene and/or the LTC4S gene, and correlations betweenthe polymorphic allele genotype and the occurrence of a phenotypicresponse in the population of subjects are determined. Detection of agenotype that is correlated with an increased or decreased incidence ofa desired therapeutic response or a side effect (compared to theincidence in subjects with alternative genotypes) indicates that theeffectiveness of the ligand in treating the respiratory disease variesamong genetic subpopulations.

Clinical trials of the type discussed in this application generatevarious kinds of data that is advantageously stored on electronicallyreadable media, including, but not limited to magnetic tapes, magneticdisks, solid state memory and storage devices, optically readable disksand any combination of these. Such data is also advantageouslytransmitted or communicated via telecommunications means includingmetallic or optical fiber lines or via wireless electromagneticfrequency devices. Additionally, such data is advantageouslycommunicated via at least two or more electronic computing devices,including personal computers, computer workstations, computer servers,mainframe computers, super computers and the like. Such communicationscan occur either directly from device to device or through a pluralityof such devices that have been electronically instructed on how to routesuch communications from a sender to a designated receiver of suchcommunication, including, but not limited to, organizational intranetsand the internet or the world wide web. Such data that can be stored andcommunicated in the above ways include, but are not limited to, anynucleotide sequence data, amino acid sequence data, protein-proteininteraction data, clinical diagnosis data or statistics data generatedby the above clinical trials. The use of such electronic devices asdescribed is an alternative embodiment of the invention claimed herein,particularly when used commercially.

Additionally, the present invention affords a way of designing andconducting clinical trials in such a way as to take advantage of thosepatients who do not respond to a leukotriene receptor antagonist, byre-testing that population in a way calculated to increase thelikelihood of discovering therapies for such non-responding patients.Presently, the marketing of novel medicines requires extensive clinicaltrials conducted to demonstrate the efficacy and safety of the candidatemedicine. In any given clinical trial, there will be an observedpercentage of the patients enrolled in that trial who will eitherrespond to the candidate medicine as hoped for, or who will respond lessstrongly, or who will not respond at all. Also, there are three possibleadverse side effect outcomes, namely no adverse side effect, someacceptable degree of adverse side effect, and an unacceptable adverseside effect. Currently available data suggest that a major part of thepartial responders or non-responders populations results from multipleetiologies leading to the recognized phenotype. Single nucleotidepolymorphism (SNP) profiling of different medicine-responsiveassociation groups during such clinical studies implies that thelocation of genes contributing to heterogeneous forms of the disease canalso be identified, leading to the discovery of additionalsusceptibility targets.

A systematic study of those populations of patients who respond to agiven medicine is complemented by a study of the converse, that is, asystematic study of those subgroups of patients who did not respond withefficacy and acceptable safety outcomes to the initially studiedmedicine. Such partial responders or non-responders could be identifiedin real time, rather than by the current trial and error system thatperforms this function once a medicine appears in the marketplace.Currently drugs are broadly marketed to many patients who will notbenefit and who may experience adverse reactions. Little information ofuse is obtained from these patients currently, other than broad warningsfor use in product labeling.

There is thus a need in the art for a method of designing human clinicaldrug trials in a fashion that benefits available patient populationsthrough 1) minimizing the likelihood of adverse events, 2) maximizingthe likelihood of therapeutic response and 3) providing a pool of datathat readily suggests a subsequent clinical trial that is capable ofutilizing all prior data, whether for responders or for non-responder.The present invention therefore additionally includes a method ofconducting clinical drug trials by pharmacogenetic stratification of apatient population, comprising the steps of conducting a first clinicaldrug trial on a patient population, such that said drug trial identifiesan association between a phenotype (response or non-response to aleukotriene antagonist) and a genotype; separating said patientpopulation in said clinical drug trial into sub-populations ofresponders and non-responders; conducting a subsequent clinical drugtrial on a non-responder patient population such that said subsequentdrug trial identifies a subsequent association between a phenotype and agenotype; separating the patient population of step (c) into subsequentresponder and subsequent non-responder patient populations; and thenrepeating steps (c) and (d) through as many iterations as desired.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing the results of a previous study of thecorrelation between Sp1 polymorphisms at the ALOX5 gene alone anddiminished response to the leukotriene synthesis inhibitor compoundABT-761 (Abbott Laboratories Inc., North Chicago, Ill.). The study was arandomized double-blind parallel-group trial (n=221) of clinicallystable asthma patients, with FEV₁ of 40%-75% of predicted. ABT-761 wasgiven at 150 and 300 mg/day. The vertical axis graphs the percent changein baseline FEV₁ after drug treatment; the horizontal axis providesgenotypes (5,5=homozygous wild-type; 5,X=heterozygous wild-type; X,X=twonon-wildtype alleles). Approximately 6% of the subjects were without thewild-type allele. The change in the variant group was significantlydifferent from the 5,5 group (*p<0.0001).

FIG. 2 is a graph showing the correlation between Sp1 polymorphisms atthe ALOX5 gene and response to the leukotriene inhibitor compoundzafirlukast, also showing the relative difference in such response ascompared to fluticasone propionate (FP88). The vertical axis graphs thechange in baseline FEV₁ (% predicted, change from baseline) after drugtreatment; the horizontal axis provides genotypes (5,5=homozygouswildtype; 5,X=heterozygous wildtype; X,X=two non-wildtype alleles); openbars represent fluticasone propionate; shaded bars representzafirlukast. Of the subjects, 88 (59.5%) had genotype 5,5 and 44 eachwere treated with FP88 and zafirlukast; 50 (33.8%) were 5,X (31 treatedwith FP88 and 19 treated with zafirlukast); and 10 (6.8%) were X,X (5treated with FP88 and 5 treated with zafirlukast).

FIG. 3 is a graph showing the correlation between polymorphisms at theA-444C site in the LTC4 Synthase gene and response to the leukotrieneinhibitor compound zafirlukast, also showing response to fluticasonepropionate (FP88). The vertical axis graphs the change in baseline FEV₁(% predicted, change from baseline) after drug treatment; the horizontalaxis provides genotypes (A/A=homozygous wildtype; A/C=heterozygouswildtype; C/C=homozygous for adenine to cytosine transversionpolymorphism); open bars represent fluticasone propionate; shaded barsrepresent zafirlukast. Asterisks indicate a significant difference(p≦0.05) between FP88 and zafirlukast results within a genotype. Of thesubjects, 82 (57.8%) were A,A (48 treated with FP88, 34 treated withzafirlukast), 50 (35.2%) were A,C (20 treated with FP88, 30 treated withzafirlukast), 10 (7%) were C,C (6 treated with FP88, 4 treated withzafirlukast).

FIG. 4 is a graph showing the correlation between A1728G polymorphismsin the ALOX5 gene and response to fluticasone proprionate (FP88) andzafirlukast. The vertical axis represents the change in baseline FE₁ (%predicted, change from baseline) after drug treatment; the horizontalaxis provides genotypes (A/A=homozygous wildtype; A/G=heterozygouswildtype; G/G=homozygous for adenine to guanine transversionpolymorphism); open bars represent fluticasone propionate; shaded barsrepresent zafirlukast. Of the subjects, 130 were A,A (72 treated withFP88, 58 treated with zafirlukast), 18 were A,G (8 treated with FP88, 10treated with zafirlukast), and 1 was G,G (treated with zafirlukast).

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have determined that polymorphic variations in theALOX5 gene and/or the LTC4S gene can be correlated to the response topharmaceutical treatment, particularly treatment with leukotrienereceptor antagonists, more particularly with CysLT1 leukotriene receptorantagonists, and also particularly with glucocorticoids. The presentinventors have identified that there exists a single nucleotidepolymorphism in the promoter region of enzyme LTC4 Synthase; an adenineto cytosine transversion 444 nucleotides upstream from the first codon,that is correlated with the response of subjects with asthma totreatment with a leukotriene receptor antagonist. They have furtheridentified polymorphisms in the ALOX5 gene which are correlated withpatient response. One such ALOX5 polymorphism is in the promoter regionand contains 3 to 6 tandem repeats of the Sp-1 binding motif GGGCGG;individuals homozygous for the most commonly occurring allele (fivetandem Sp1-binding motifs) are wild type, and individuals having 3, 4 or6 tandem repeats of the Sp-1 binding motif are considered variant (i.e.,non-wildtype). (Other numbers of tandem repeats may be found to bevariant). Additional ALOX5 polymorphisms investigated include a G to Atransversion polymorphism 1708 bases upstream from the first codon ofthe ALOX5 gene (G-1708A), and a single base transversion polymorphism1728 bases downstream from the first codon (in exon 13) of the ALOX5gene (A1728G or Pro576Pro). ALOX5 polymorphisms have also been found tobe correlated with the response of subjects with asthma to treatmentwith a leukotriene receptor antagonist.

Little is known about the functionality of the LTC4 synthase promoterpolymorphism, although an association with this allele and aspirinintolerant asthma has been published (Sanak, M et al Leukotriene C4synthase promoter polymorphism and risk of aspirin-induced asthma. TheLancet 350;1599-1600, 1997). Applicants found a frequency of 57.6%homozygous wild type, 34.7% heterozygous, and 7.6% homozygous variantfor this LTC4 synthase promoter polymorphism. These frequencies aresimilar to those found with the ALOX5 promoter polymorphism, yet thegenes for these enzymes reside on different chromosomes.

As used herein, a “respiratory disorder that is potentially treatable”with a particular compound is one in which such treatment has been shownto be beneficial in a significant number of subjects suffering from thatrespiratory disorder. As will be apparent to those skilled in the art, acondition that is treatable with a certain compound or class ofcompounds does not imply that every patient so treated will benefit.

Polymorphisms may be present in the coding sequence of a gene, or in the5′ or 3′ noncoding regions. The 5′ noncoding region includes sequencesimportant in transcriptional regulation, including promoters, negativeregulatory regions, and positive regulatory regions.

Genetic samples were obtained from subjects enrolled in clinical trialsfor the treatment of asthma. The genetic samples were first screened foran adenine to cytosine transversion polymorphism 444 nucleotidesupstream from the first codon in the leukotriene C4 Synthase gene (LTC4Sgene), using polymerase chain reaction (PCR) technology. The alleleswere labeled as “A” (adenine) or “C” (cytosine) resulting in threepossible genotypes (A/A, A/C, and C/C

The nucleotide sequence of human leukotriene C4 synthase (LTC4S) isprovided in GenBank at accession number U50136 (SEQ ID NO:1):

1 gagctcacag agcccccagc tggggcatat ctggtttccg ggggcagggg cgatacccag 61aggaggaaga agggattctg agagagccca acaggctccg agcctcaggc tggagctgag 121cttggggcag caaggaagga ccaggtgcga gggcagaacc atgcggcccg acccctgcag 181cacggcctgt ggcctccccc agctcctgcc cgtgcttctg ggtcagtctg gactttgcca 241cttctgacca aaagccaccg caaacccact caagccaaaa gaggaagtga ccgttaggcc 301caactgggaa ggctggcggc caggggcact ccaggcaggg cgaggggggc ggccgggggc 361gctccaggcg gggcgaggga gacacccaga actccaggca ggagtcctcg ggtgccacct 421ttcctctcca cctggccctg cgtgggctct gtcctcaggg tggcccgccg tagtccccct 481ccccactctg agtttcctgt cccaaagtcc taaggaagtt tccagaacta catctcacca 541tcttgagtca gccttggctc agtgtccatc tcacaggcct ggaaggggca ggagtcagca 601ctgtccagac cacagggcct gagtgtgggg agggcagccg tctaggaagg tggtggaggg 661ttgttacctt gaggcaagag ggctgcgggg cagaaagaca cagcaggtga ctgttgtggg 721aggcccaaga gaggcctggg agagggatgg cccacaaggg ctgaccctcc cgccacccag 781ggggccttgg acaggtttcc tcctggcagg gtggcccttg tgcatggaac ccctacaacg 841actaaggctg gcaggcatga ggtttcctga aggagaaaga gcttgtgggg cccagtgtgg 901ctgggggggc gctgggactc cattctgaag ccaaaggcac tgggaagggc ttccgcagag 961gagggtttgg caggggttgc caggaacagc ctggatgggg aca gggaaca gataaggtgg 1021gtggaggagt tagccgggag cctggggctg gctccagcat gatgtggggg tctgcaaggc 1081cctggagaaa gtggggtggt gcagcagggg gcacacccac agctggagct gacccagatg 1141gacagcttgg gctctgccac gcgggactag gcaaggaagg ggcacgaaca agcaggaagt 1201ggtgaggcgg tctccagcta gctgctctcc cctgcccaga ctttggtttc ctccctgctg 1261gcttggcctg gctccctggc tctgtgtggt atggtcacac ccccgtgcac cccctccact 1321gagatggggc ggggagagca ccgaggctgc tcttcctctc ctgggccgtc ctctgagcag 1381cagacggggc taagcgttcc ccagctcgcc ttcacacaca gcccgtgcca ccacaccgac 1441ggtaccatga aggacgaggt agctctactg gctgctgtca ccctcctggg agtcctgctg 1501caaggtgggc tggttcctat ctaggaagag ggtgggcctt agatccctac agcttgccct 1561ctgcccccta ggcccaggtg gagggcagag gtggggactc cagcccaggc ccaagctgga 1621agagggtggg gactttcagg gaactggggg gcacctggct gtgagagctg taggacttgg 1681gggtggcaag ggtgccagga caaatggtag gatagccatg ggcttgggga agctgatctc 1741tgctctttcc agctgtcccc tctctgggcg tcccagcaag cggcccccat tccctggctc 1801tgcttcaaag gcacctccat actgggacca cgtggagcag ggtagaggtg ggactccttc 1861ctccagcccc ctaaaaagag cctgcttaat gcctttctca gactggccct aaaggacaca 1921ttccttggcc agatatcctt gccacctaag agacaccact actccacagt gtgtgggcta 1981ggataaggca cagcctgggg agggggctct gaaggggctg aacagacagg ccagcctgac 2041ctccagctgc tcctgcactg agctggatgg ccaccctgtg acacccatct gcagagggcc 2101cagaaccaaa ggtgccaggg ctgcaggact cagggggaga tggtccgacg ggaggtctgg 2161ggagggagcg cacagccagc actggtctgt gtgtggtctg gcctggcctc acctgaccaa 2221gagaagggct cctgcccaca gagaaacttt agggccagcc caccctctgc aactacccca 2281gccctggggt cctggggtta ggctaggaga gtcccagctg caacctcctg ggagcaggag 2341agaaggtgtc tgtcagattt aggcctggga ccggaatgca ggaacagaga aactgaggtt 2401tggaggcaca gggacgcagg ctttagtgat cccggcctga ggcagggtca gagggccctg 2461ctggtgggcg ctggtaggtg ggtgaccagg gactgttagc tacagggagt gtgcttcctt 2521gcacctggga ggatgcagcc agctctgccc tcagactccc gaggcacttc ctggccaggg 2581acctgaaagc tgcatttgcc tgtgttttga gagtgaaatg attcagaaac aaggactcaa 2641gtggtctctc tcgcggagca ggtgtccctg tgcctgaatc actcaccctc ccccatacac 2701tcacaggttg ggacagggcc tctctgcgcc ccaggcttca gccctgccct cctcgctgaa 2761tgtcagggac acagggcagg ccagggatgg gtgagacgag aggtctcctc gggcggggag 2821ggggcggggt tccgccttag ggaggagagg acacggccaa gtgaagggcc agattgcagg 2881atccctccca ctcccatctc tggggcttcg ggtgtccaga cctgactccc gctccccctc 2941ctcccccagc ctacttctcc ctgcaggtga tctcggcgcg cagggccttc cgcgtgtcgc 3001cgccgctcac caccggccca cccgagttcg agcgcgtcta ccgagcccag tgaggcgcgg 3061cgggagggcg cggggcgggg agcgagcccc aggcgggtcc gggtcgcagg accatcccgg 3121ccggcgcgct catcccaccc gcccaccgca gggtgaactg cagcgagtac ttcccgctgt 3181tcctcgccac gctctgggtc gccggcatct tctttcatga aggtcggggt gtggggcagg 3241ggcgcacgcg ctggaccccc gggacccgcg cagggcgctc accaggcccg tgcgtacctc 3301tcgcaggggc ggcggccctg tgcggcctgg tctacctgtt cgcgcgcctc cgctacttcc 3361agggctacgc gcgctccgcg cagctcaggt gagggccggg cggggagcgg ggcggggccg 3421gggaaagatc gcgggcgggc ggggctcctg gggagcggga ccgaagctgg gggcgggcga 3481cgggccggag cccagcgcct ttggggattc ggtgggcgag ccctggcggc ggccagagga 3541agtccccgtg gggccagggt tgcggcgggg aagaagcggg cctcctcgcg ccacctcccc 3601gctgaccgcc gcccgcaggc tggcaccgct gtacgcgagc gcgcgcgccc tctggctgct 3661ggtggcgctg gctgcgctcg gcctgctcgc ccacttcctc ccggccgcgc tgcgcgccgc 3721gctcctcgga cggctccgga cgctgctgcc gtgggcctga gaccaaggcc cccgggccga 3781cggagccggg aaagaagagc cggagcctcc agctgccccg gggaggggcg ctcgcttccg 3841catcctagtc tctatcatta aagttctagt gaccgagacc cgggctgcgt tctctgggtc 3901cgcgggggtg gcgcaccgcg ggctacggag cctggagggg cccagcccga gtccgggcag 3961cccggggcgg gcttcctagt ggcggcgtga gagtggctgc gaaggaacga gccctccccc 4021tggggcggga ctggatccgg tcttcacctc ctaccccact ccctactcag cctcggggtc 4081acaaggccgc ccagtcctgc cggggttcac cctcctagcg ctcagcggtc tcctcaccgg 4141tccccctcct caggggcctt ccctcgactc tcagccgccg cagtccctcg tcccctggcc 4201ttcacagctg acactagata gagcctgtgg ctctctcccc aggtgagggc aggggttttt 4261cttttggtca gcactggatc cccctcgtta actgtaggtg ttcagggcag ccctccgagg 4321tccgcagagc tgcgggcacc atgggaacga agtgagtcag tgacaggcgg tctcaaggaa 4381atgtccagaa gccttgggga tccaggggag gcccacagaa acaaagaagt gacttttagc 4441caagtatgca ggagaaacgg aggagThe protein encoded by the above is provided at SEQ ID NO:2.

An adenine to cytosine transversion polymorphism (A-444C) is known atthe position 444 nucleotides upstream (bold and underlined, above) fromthe first codon (underlined, above). Accordingly, the “A” allele asdefined herein would comprise the sequence CTGGATGGGG ACAGGGAACA (SEQ IDNO:3) (nucleotides 991-1010 of SEQ ID NO:1). In contrast, the “C” alleleas defined herein would comprise the sequence CTGGATGGGG ACCGGGAACA (SEQID NO:4) (nucleotides corresponding to 991-1010 of SEQ ID NO:1).

The genetic samples were secondly screened for a polymorphism in theregion of 176 to 147 base pairs upstream from the ATG start site in5-lipoxygenase (ALOX5), whereby the presence of 5 tandem repeats of theSp1 binding motif (GGGCGG; SEQ ID NO:12) is wildtype, and the presenceof 3, 4 or 6 such tandem repeats is variant, again using PCR technology.The alleles were labeled as “5” (5 tandem repeats) or “X” (3, 4 or 6tandem repeats), resulting in three possible genotypes (5,5, 5/X andX/X). Variant ALOX5 alleles have a number of tandem repeats of the Sp1binding motif in this region that is more than, or fewer than, fiverepeats. That is, in the region of from about 200 nucleotides to about125 nucleotides upstream of the ATG start site in ALOX5, there are fromone to four, or more than five, repeats of the Sp1 binding motif. In thepresent studies, variant ALOX5 alleles (“X”) had three, four or sixtandem repeats in this area.

Screening was also conducted for a G to A transversion polymorphism 1708bases upstream from the first codon of the ALOX5 gene (G-1708A), and fora single base transversion polymorphism 1728 bases downstream from thefirst codon (in exon 13) of the ALOX5 gene (A1728G).

The nucleotide sequence of the 5′ region and partial coding sequence ofhuman 5-lipoxygenase (ALOX5) containing five repeats of the Sp1 bindingmotif, and the G-1708A polymorphism, is provided at GenBank Accessionnumber M38191(SEQ ID NO:5):

1 ggatccagaa taaccaaaac aatattgaaa aataaagaac agcgttggtg gattaacatt 61ttccaatttc aaaacttact atagcactgc ggtaatcaag cagtgtggca ctgtatagca 121tgtacattac agatcagtgg actagaatca atgtccagaa ataaaccgtt atgtttataa 181tgaattactt tttaataagg tgtcaagaca acgcaatggg aaaagaataa tgaattcaac 241aaatgatgca tggacaaccg gacatgcaca tgcaacacaa tgaatttgaa ttcttctatc 301gctccatgca taaaaactaa ctcaaaatgg gtcacggatg taaatgaaaa gctaaaacta 361taataatcct agaggaaaac ctaggagtaa atctttaaga tgttattgta ggcagtggtt 421tctcagatag gaccccaaaa tcacaagcga caaaaagaaa ttggacttaa agttaaatac 481ttttgtgctt caaacatcat caagaaagtg aaaacacaac ccgcagaagc aataaaaatg 541tctgtaagtc atgtatccga ttagagactt ctatccagga tatataaata atgcaattca 601atgataaaaa agataaatag cccagttttc caaagagtca agcatctgaa tatacatctc 661tccaaaaata tacagatatc caacaagcat gtgaaaagat gttcaaagcc atttgccagg 721tgcacaaacc caagacagta tgaggagatg ctacagggac tctgctgctt cacagacatg 781aagcgttggt gagaatgtag gcagccgcct ttggggactt cacatccccg ccgccccacg 841cacggtgagc tagtgtttaa acttagccga gatcaataca cgcgactgtg tgcccgtcag 901accctgcgct gccggcgggg ctgggagagg cgggcgccag gagtgggcgg gaacctgggg 961gtcaggcccc agccgcggga agcgcgccca ggagcgcgcg aaaccttctc cacacccttc 1021caggcatttg cccgccgcga ttcagagagc cgacccgtga cccctggcct cccctagaca 1081gccccgcatg tccagatgtg ccgtcccgcc tgcctcccgc gaccactggc catctctggg 1141cctgggcgcg gttctcggcg cccggcctgc ccccgccagg agccgcaggt ccagccagtg 1201aagaagcccg cgcctgaagg agcctctgtg ctccagaatc catcctcagt atcagcgctg 1261gggtggcctc ctccaggaag cccttctgat tctctcatgg gtcgctcttc ctctgcagac 1321tcccggagca ccccctgctc caagtaccgc aagtggcact gagaacttgg ggagagcaga 1381ggctgtgcct agatttgtag ggagtccccg cagctccacc ccagggccta caggagcctg 1441gccttgggcg aagccgaggc aggcaggcag ggcaaagggt ggaagcaatt caggagagaa 1501cgagtgaacg aatggatgag gggtggcagc cgaggttgcc ccagtcccct ggctgcagga 1561acagacacct cgctgaggag agacccagga gcgaggcccc tgccccgccc gaggcgaggt 1621cccgcccagt cggcgccgcg cgtgaagagt gggagagaag tactgcggg g g c gggggcgg1681 gggcgggggc gggggcgggg gcagccggga gcctggagcc agaccggggc ggggccggga1741 ccggggccag ggaccagtgg tgggaggagg ctgcggcgct agatgcggac acctggaccg1801 ccgcgccgag gctcccggcg ctcgctgctc ccgcggcccg cgccatgccc tcctacacgg1861 tcaccgtggc cactggcagc cagtggttcg ccggcactga cgactacatc tacctcagcc1921 tcgtgggctc ggcgggctgc agcgagaagc acctgctgga caagcccttc tacaacgact1981 tcgagcgtgg cgcggtgagc gcgggcgggg cacgggtgga gcgcgggctg aggtgcgtcc2041 gggacccggt ttggacggca gaggcctggg cgggggcgcc gagggcccgt cggggcggcc2101 cggacaggac tgggggtgtc caggaccctg tcagggaggg cagaactgcg gtggggcgtg2161 ccctgggctc ccagtggccg gtgggtaccThe first codon is underlined; the region comprising the repeats of theSp1 binding motif (GGGCGG) is shown in bold and underlined type; theG-1708A position is also shown in bold and underlined type.

The 5-lipoxygenase gene has been cloned as cDNA (Matsumoto et al., Proc.Natl. Acad. Sci. USA 85:3406 (1988) and as a genomic clone (Hoshiko etal., Proc. Natl. Acad. Sci. USA 87:9073 (1990). The 5-lipoxygenase geneis approximatly 85 kilobases in size, with 14 exons and 15 introns.

Two ALOX5 mRNA sequences are provided in GenBank at Accession numbers NM000698 and XM 005818. The sequence provided at NM 000698 encodes aprotein of 674 amino acids and is shown below:

(SEQ ID NO:13) 1 gggcgccgag gctccccgcc gctcgctgct ccccggcccg cgccatgccctcctacacgg 61 tcaccgtggc cactggcagc cagtggttcg ccggcactga cgactacatctacctcagcc 121 tcgtgggctc ggcgggctgc agcgagaagc acctgctgga caagcccttctacaacgact 181 tcgagcgtgg cgcggtggat tcatacgacg tgactgtgga cgaggaactgggcgagatcc 241 agctggtcag aatcgagaag cgcaagtact ggctgaatga cgactggtacctgaagtaca 301 tcacgctgaa gacgccccac ggggactaca tcgagttccc ctgctaccgctggatcaccg 361 gcgatgtcga ggttgtcctg agggatggac gcgcaaagtt ggcccgagatgaccaaattc 421 acattctcaa gcaacaccga cgtaaagaac tggaaacacg gcaaaaacaatatcgatgga 481 tggagtggaa ccctggcttc cccttgagca tcgatgccaa atgccacaaggatttacccc 541 gtgatatcca gtttgatagt gaaaaaggag tggactttgt tctgaattactccaaagcga 601 tggagaacct gttcatcaac cgcttcatgc acatgttcca gtcttcttggaatgacttcg 661 ccgactttga gaaaatcttt gtcaagatca gcaacactat ttctgagcgggtcatgaatc 721 actggcagga agacctgatg tttggctacc agttcctgaa tggctgcaaccctgtgttga 781 tccggcgctg cacagagctg cccgagaagc tcccggtgac cacggagatggtagagtgca 841 gcctggagcg gcagctcagc ttggagcagg aggtccagca agggaacattttcatcgtgg 901 actttgagct gctggatggc atcgatgcca acaaaacaga cccctgcacactccagttcc 961 tggccgctcc catctgcttg ctgtataaga acctggccaa caagattgtccccattgcca 1021 tccagctcaa ccaaatcccg ggagatgaga accctatttt cctcccttcggatgcaaaat 1081 acgactggct tttggccaaa atctgggtgc gttccagtga cttccacgtccaccagacca 1141 tcacccacct tctgcgaaca catctggtgt ctgaggtttt tggcattgcaatgtaccgcc 1201 agctgcctgc tgtgcacccc attttcaagc tgctggtggc acacgtgagattcaccattg 1261 caatcaacac caaggcccgt gagcagctca tctgcgagtg tggcctctttgacaaggcca 1321 acgccacagg gggcggtggg cacgtgcaga tggtgcagag ggccatgaaggacctgacct 1381 atgcctccct gtgctttccc gaggccatca aggcccgggg catggagagcaaagaagaca 1441 tcccctacta cttctaccgg gacgacgggc tcctggtgtg ggaagccatcaggacgttca 1501 cggccgaggt ggtagacatc tactacgagg gcgaccaggt ggtggaggaggacccggagc 1561 tgcaggactt cgtgaacgat gtctacgtgt acggcatgcg gggccgcaagtcctcaggct 1621 tccccaagtc ggtcaagagc cgggagcagc tgtcggagta cctgaccgtggtgatcttca 1681 ccgcctccgc ccagcacgcc gcggtcaact tcggccagta cgactggtgctcctggatcc 1741 ccaatgcgcc cccaaccatg cgagccccgc caccgactgc caagggcgtggtgaccattg 1801 agcagatcgt ggacacgctg cccgaccgcg gccgctcctg ctggcatctgggtgcagtgt 1861 gggcgctgag ccagttccag gaaaacgagc tgttcctggg catgtacccagaagagcatt 1921 ttatcgagaa gcctgtgaag gaagccatgg cccgattccg caagaacctcgaggccattg 1981 tcagcgtgat tgctgagcgc aacaagaaga agcagctgcc atattactacttgtccccag 2041 accggattcc gaacagtgtg gccatctgag cacactgcca gtctcactgtgggaaggcca 2101 gctgccccag ccagatggac tccagcctgc ctggcaggct gtctggccaggcctcttggc 2161 agtcacatct cttcctccga ggccagtacc tttccattta ttctttgatcttcagggaac 2221 tgcatagatt gtatcaaagt gtaaacacca tagggaccca ttctacacagagcaggactg 2281 cacaggcgtc ctgtccacac ccagctcagc atttccacac caagcagcaacagcaaatca 2341 cgaccactga tagatgtcta ttcttgttgg agacatggga tgattattttctgttctatt 2401 tgtgcttagt ccaattcctt gcacatagta ggtacccaat tcaattactattgaatgaat 2461 taagaattgg ttgccataaa aataaatcag ttcattt

The 1728 polymorphism site is indicated in underlined and bolded type;the A1728G polymorphism does not change the encoded amino acid (prolineat amino acid position 576; SEQ ID NO: 14).

The sequence at XM 005818 is provided below (SEQ ID NO:15) and encodesan amino acid sequence (SEQ ID NO:16) that differs from that encoded byNM000698 in the initial amino acids. The site of the A1728G polymorphism(numbering referenced to NM000698) is shown in underlined bold type.

(SEQ ID NO:15) 1 cttcaccccg tggtgaagac actgacgact acatctacct cagcctcgtgggctcggcgg 61 gctgcagcga gaagcacctg ctggacaagc ccttctacaa cgacttcgagcgtggcgcgg 121 tggattcata cgacgtgact gtggacgagg aactgggcga gatccagctggtcagaatcg 181 agaagcgcaa gtactggctg aatgacgact ggtacctgaa gtacatcacgctgaagacgc 241 cccacgggga ctacatcgag ttcccctgct accgctggat caccggcgatgtcgaggttg 301 tcctgaggga tggacgcgca aagttggccc gagatgacca aattcacattctcaagcaac 361 accgacgtaa agaactggaa acacggcaaa aacaatatcg atggatggagtggaaccctg 421 gcttcccctt gagcatcgat gccaaatgcc acaaggattt accccgtgatatccagtttg 481 atagtgaaaa aggagtggac tttgttctga attactccaa agcgatggagaacctgttca 541 tcaaccgctt catgcacatg ttccagtctt cttggaatga cttcgccgactttgagaaaa 601 tctttgtcaa gatcagcaac actatttctg agcgggtcat gaatcactggcaggaagacc 661 tgatgtttgg ctaccagttc ctgaatggct gcaaccctgt gttgatccggcgctgcacag 721 agctgcccga gaagctcccg gtgaccacgg agatggtaga gtgcagcctggagcggcagc 781 tcagcttgga gcaggaggtc cagcaaggga acattttcat cgtggactttgagctgctgg 841 atggcatcga tgccaacaaa acagacccct gcacactcca gttcctggccgctcccatct 901 gcttgctgta taagaacctg gccaacaaga ttgtccccat tgccatccagctcaaccaaa 961 tcccgggaga tgagaaccct attttcctcc cttcggatgc aaaatacgactggcttttgg 1021 ccaaaatctg ggtgcgttcc agtgacttcc acgtccacca gaccatcacccaccttctgc 1081 gaacacatct ggtgtctgag gtttttggca ttgcaatgta ccgccagctgcctgctgtgc 1141 accccatttt caagctgctg gtggcacacg tgagattcac cattgcaatcaacaccaagg 1201 cccgtgagca gctcatctgc gagtgtggcc tctttgacaa ggccaacgccacagggggcg 1261 gtgggcacgt gcagatggtg cagagggcca tgaaggacct gacctatgcctccctgtgct 1321 ttcccgaggc catcaaggcc cggggcatgg agagcaaaga agacatcccctactacttct 1381 accgggacga cgggctcctg gtgtgggaag ccatcaggac gttcacggccgaggtggtag 1441 acatctacta cgagggcgac caggtggtgg aggaggaccc ggagctgcaggacttcgtga 1501 acgatgtcta cgtgtacggc atgcggggcc gcaagtcctc aggcttccccaagtcggtca 1561 agagccggga gcagctgtcg gagtacctga ccgtggtgat cttcaccgcctccgcccagc 1621 acgccgcggt caacttcggc cagtacgact ggtgctcctg gatccccaatgcgcccccaa 1681 ccatgcgagc cccgccaccg actgccaagg gcgtggtgac cattgagcagatcgtggaca 1741 cgctgcccga ccgcggccgc tcctgctggc atctgggtgc agtgtgggcgctgagccagt 1801 tccaggaaaa cgagctgttc ctgggcatgt acccagaaga gcattttatcgagaagcctg 1861 tgaaggaagc catggcccga ttccgcaaga acctcgaggc cattgtcagcgtgattgctg 1921 agcgcaacaa gaagaagcag ctgccatatt actacttgtc cccagaccggattccgaaca 1981 gtgtggccat ctgagcacac tgccagtctc actgtgggaa ggccagctgccccagccaga 2041 tggactccag cctgcctggc aggctgtctg gccaggcctc ttggcagtcacatctcttcc 2101 tccgaggcca gtacctttcc atttattctt tgatcttcag ggaactgcatagattgatca 2161 aagtgtaaac accataggga cccattctac acagagcagg actgcacagcgtcctgtcca 2221 cacccagctc agcatttcca caccaagcag caacagcaaa tcacgaccactgatagatgt 2281 ctattcttgt tggagacatg ggatgattat tttctgttct atttgtgcttagtccaattc 2341 cttgcacata gtaggtaccc aattcaatta ctattgaatg aattaagaattggttgccat 2401 aaaaataaat cagttcattt

The present inventors have determined that the C/C genotype at theA-444C site of LTC4S, the X/X Sp1 genotype in ALOX5, and thepolymorphism at the A1728G site of ALOX5 are independently associatedwith diminished response to the Cys LT1 leukotriene receptor antagonistzafirlukast.

According to the present methods, a subject who suffers from asthma thatis potentially treatable with an anti-inflammatory inhaledglucocorticoid or a leukotriene antagonist, is genetically screened, toaid in predicting their response to such treatment. Screening comprisesobtaining a sample of DNA from the subject and screening the DNA todetermine the genotype (presence/absence of polymorphic alleles) at apredetermined polymorphic site in the gene of interest (here ALOX5and/or LTC4S polymorphisms as described), where different genotypes atthat site have previously been associated with different incidences of aphenotypic response to treatment. The presence of a particular genotypetherefore indicates an increased likelihood that the individual subjectwill exhibit the associated phenotype. The genotype will rarely beabsolutely predictive, i.e., where a population with a certain genotypedisplays a high incidence of a particular phenotype, not everyindividual with that genotype will display the phenotype. However, itwill be apparent to those skilled in the art that genotyping a subjectas described herein will be an aid in predicting the response a subjectwill have to treatment with a leukotriene receptor antagonist or aglucocorticoid, and thus assist in the treatment decision.

As used herein, “genotyping a subject (or DNA sample) for a polymorphicallele at a defined genomic locus” or “determining the genotype at apolymorphic allelic site” means detecting which forms of the allele arepresent in a subject (or a biological sample). As is well known in theart, an individual may be heterozygous or homozygous for a particularallele. More than two forms of an allele may exist, as is the case withmicrosatellite markers; thus there may be more than three possiblegenotypes.

As used herein, a subject that is “predisposed to” a particularphenotypic response based on genotyping of a polymorphic allele will bemore likely to display that phenotype than an individual with adifferent genotype at that polymorphic allele. Where the phenotypicresponse is based on a biallelic polymorphism, the response may differamong the three possible genotypes (Eg. For LTC4S: A,A; A,C and C,C).

As used herein, a “genetic subset” of a population consists of thosemembers of the population having a particular genotype. In the case of abiallelic polymorphism, a population can potentially be divided intothree subsets: homozygous for allele 1, heterozygous, and homozygous forallele 2. Where multiple non-wildtype polymorphisms exist, a populationcan also be divided into three subsets: homozygous wildtype;heterozygous wildtype; and homozygous non-wildtype.

As used herein, asthma treatable with an anti-inflammatoryglucocorticoid or treatable with a leukotriene receptor antagonist is adisease in which the administration of such a drug (in an appropriatepharmaceutical formulation, and in a therapeutically effective amount)has been shown to reduce or alleviate symptoms, without causingunacceptable side effects. Such therapeutic effectiveness is typicallyevidenced by Regulatory Authority (eg FDA, EMEA) approval of thepharmaceutical preparation, or by publication of the results of clinicalstudies in peer-reviewed medical journals. Therapeutically effectiveamounts of such compounds can be readily determined by those skilled inthe art using, e.g., dose-response studies.

Known leukotriene receptor antagonists include zafirlukast, montelukast,pranlukast or iralukast.

As used herein, a “side effect” is an undesirable response to theadministration of a therapeutic compound, e.g., an effect that is notdirected to alleviating the symptoms or cause of the disease beingtreated. Side effects range from minor inconveniences to more seriousevents.

As used herein, “response” to treatment with a therapeutic compound is adesirable response to the administration of the compound, e.g.,alleviation of the symptoms of the disease or of the underlyingpathologic causes of the symptoms. Various indicators of a subject'sresponse to therapeutic treatment may be assessed, as will be apparentto one skilled in the art. As an example, the change in ForcedExpiratory Volume (FEV; FEV₁=FEV for 1 second duration) may be used asan indicator of response to treatment for asthma, as will be apparent toone skilled in the art.

According to the present methods, a compound with leukotriene receptorantagonism may be screened for variation in its effects among geneticsubpopulations of subjects with asthma. Such methods involveadministering the compound alone, or in tandem with another anti-asthmacompound (such as a glucocorticoid), to a population of subjectssuffering from asthma, obtaining DNA samples from the subjects (whichmay be done either prior to or after administration of the compounds),genotyping a polymorphic allelic site in the gene of interest, andcorrelating the genotype of the subjects with their phenotypic responses(both favorable and unfavorable) to the treatment. A genotype that iscorrelated with an increased incidence of a desired therapeuticresponse, compared to the incidence in subjects with alternativegenotypes at the polymorphic allelic site, indicates that theeffectiveness of the compound in treating asthma varies among geneticsubpopulations.

Stated another way, the methods of the present invention may be used todetermine the correlation of a known polymorphic allele with theresponse of subjects to treatment with a leukotriene receptor antagonistor a glucocorticoid. The population of subjects with the disease ofinterest is stratified according to genotype for the particularpolymorphic allele, and their response to a therapeutic agent isassessed (either prospectively or retrospectively) and compared amongthe genotypes. The response to the therapeutic agent may include either,or both, desired therapeutic responses (e.g., the alleviation of signsor symptoms) and undesirable side effects. In this way, genotypes thatare associated with an increased (or decreased) incidence of therapeuticefficacy, or an increased (or decreased) incidence of a particular sideeffect, may be identified. The increase or decrease in response is incomparison to the other genotypes, or to a population as a whole.

Polymorphisms are variant sequences within the human genome that may ormay not have a functional consequence. These variants can be used in allaspects of genetic investigation including the analysis and diagnosis ofgenetic disease, forensics, evolutionary and population studies. Twotypes of genetic analyses are typically performed: linkage andassociation studies.

A linkage study provides genetic map information with no prior knowledgeor assumption about the function of a gene. In a linkage study one usesDNA polymorphisms to identify chromosomal regions that are identicalbetween affected relatives with the expectation that allele sharingfrequencies will be higher for a marker (polymorphism) whose chromosomallocation is close to that of the disease allele. Physical cloning of alinkage region narrows down the DNA sequence that could harbor thecandidate disease gene. While linkage analysis locates the disease locusto a specific chromosome or chromosome region, the region of DNA inwhich to search for the gene is typically large, on the order of severalmillion base pairs.

In contrast to linkage, association shows the coexistence of apolymorphism and a disease phenotype in a population. Associationstudies are based upon linkage disequilibrium, a phenomenon that occursbetween a marker and a disease loci when the occurrence of two allelesat different loci is larger than the product of the allelic frequencies.Since the marker and disease causing variant are in close proximity, itrequires many generations of recombination to separate them in apopulation. Thus they tend to co-exist together on the same chromosomeat a higher than expected frequency. A marker (polymorphism) is said tobe associated with a specific phenotype when its frequency issignificantly higher among one phenotype group compared to its frequencyin another. In general, the closer a marker is to the functionallypolymorphic site, the stronger the association.

Association studies offer the opportunity to finely map linkage regions,map loci refractory to linkage analysis and map unknown predispositionloci. Polymorphisms that are in linkage disequilibrium with each othercan be spaced over large regions. Linkage disequilibrium has beenreported in regions as small as 1 kb or as large as 500 kb.Polymorphisms throughout a gene can be in linkage disequilibrium witheach other, such that it is valuable to study the whole genomestructure—introns, exons, promoters and transcriptional regulatoryregions, and 3′ and 5′ untranslated regions. A marker that is in linkagedisequilibrium with a functional polymorphism can be tested forcorrelation with a phenotype.

As used herein, the term polymorphism includes Single NucleotidePolymorphisms (SNPs), insertion/deletion polymorphisms; transversionpolymorphisms; microsatellite polymorphisms; and variable number oftandem repeat (VNTR) polymorphisms.

Polymorphic alleles are typically detected by directly determining thepresence of the polymorphic sequence in a polynucleotide or protein fromthe subject, using any suitable technique as is known in the art. Such apolynucleotide is typically genomic DNA, or a polynucleotide derivedfrom this polynucleotide, such as in a library made using genomicmaterial from the individual (e.g. a cDNA library). The processing ofthe polynucleotide or protein before the carrying out of the method ofthe invention is further discussed below. Typically the presence of thepolymorphism is determined in a method that comprises contacting apolynucleotide or protein of the individual with a specific bindingagent for the polymorphism and determining whether the agent binds tothe polynucleotide or protein, where the binding indicates that thepolymorphism is present. The binding agent may also bind to flankingnucleotides and amino acids on one or both sides of the polymorphism,for example at least 2, 5, 10, 15 or more flanking nucleotide or aminoacids in total or on each side. In one embodiment the agent is able tobind the corresponding wild-type sequence by binding the nucleotides oramino acids which flank the polymorphism position, although the mannerof binding will be different than the binding of a polymorphicpolynucleotide or protein, and this difference will be detectable (forexample this may occur in sequence specific PCR as discussed below).

In the case where the presence of the polymorphism is being determinedin a polynucleotide it may be detected in the double stranded form, butis typically detected in the single stranded form.

The binding agent may be a polynucleotide (single or double stranded)typically with a length of at least 10 nucleotides, for example at least15, 20, 30, or more polynucleotides. The agent may be a molecule that isa structurally similar polynucleotide that comprises units (such aspurines or pyrimidines) able to participate in Watson-Crick basepairing. The agent may be a protein, typically with a length of at least10 amino acids, such as at least 20, 30, 50, 100 amino acids. The agentmay be an antibody (including a fragment of such an antibody that iscapable of binding the polymorphism).

A polynucleotide agent which is used in the method will generally bindto the polymorphism of interest, and the flanking sequence, in asequence specific manner (e.g. hybridize in accordance with Watson-Crickbase pairing) and thus typically has a sequence which is fully orpartially complementary to the sequence of the polymorphism and flankingregion.

In one embodiment of the present methods a binding agent is used as aprobe. The probe may be labeled or may be capable of being labeledindirectly. The detection of the label may be used to detect thepresence of the probe on (and hence bound to) the polynucleotide orprotein of the individual. The binding of the probe to thepolynucleotide or protein may be used to immobilize either the probe orthe polynucleotide or protein (and thus to separate it from onecomposition or solution).

In another embodiment of the invention the polynucleotide or protein ofthe individual is immobilized on a solid support and then contacted withthe probe. The presence of the probe immobilized to the solid support(via its binding to the polymorphism) is then detected, either directlyby detecting a label on the probe or indirectly by contacting the probewith a moiety that binds the probe. In the case of detecting apolynucleotide polymorphism the solid support is generally made ofnitrocellulose or nylon. In the case of a protein polymorphism themethod may be based on an ELISA system.

The present methods may be based on an oligonucleotide ligation assay inwhich two oligonucleotide probes are used. These probes bind to adjacentareas on the polynucleotide which contains the polymorphism, allowing(after binding) the two probes to be ligated together by an appropriateligase enzyme. However the two probes will only bind (in a manner whichallows ligation) to a polynucleotide that contains the polymorphism, andtherefore the detection of the ligated product may be used to determinethe presence of the polymorphism.

In one embodiment the probe is used in a heteroduplex analysis basedsystem to detect polymorphisms. In such a system when the probe is boundto a polynucleotide sequence containing the polymorphism it forms aheteroduplex at the site where the polymorphism occurs (i.e. it does notform a double strand structure). Such a heteroduplex structure can bedetected by the use of an enzyme that is single or double strandspecific. Typically the probe is an RNA probe and the enzyme used isRNAse H that cleaves the heteroduplex region, thus allowing thepolymorphism to be detected by means of the detection of the cleavageproducts.

The method may be based on fluorescent chemical cleavage mismatchanalysis which is described for example in PCR Methods and Applications3:268-71 (1994) and Proc. Natl. Acad. Sci. 85:4397-4401 (1998).

In one embodiment the polynucleotide agent is able to act as a primerfor a PCR reaction only if it binds a polynucleotide containing thepolymorphism (i.e. a sequence—or allele-specific PCR system). Thus a PCRproduct will only be produced if the polymorphism is present in thepolynucleotide of the individual. Thus the presence of the polymorphismmay be determined by the detection of the PCR product. Preferably, theregion of the primer which is complementary to the polymorphism is at ornear the 3′ end the primer. In one embodiment of this system thepolynucleotide the agent will bind to the wild-type sequence but willnot act as a primer for a PCR reaction.

The method may be an Restriction Fragment Length Polymorphism (RFLP)based system. This can be used if the presence of the polymorphism inthe polynucleotide creates or destroys a restriction site that isrecognized by a restriction enzyme. Thus treatment of a polynucleotidewith such a polymorphism will lead to different products being producedcompared to the corresponding wild-type sequence. Thus the detection ofthe presence of particular restriction digest products can be used todetermine the presence of the polymorphism.

The presence of the polymorphism may be determined based on the changethat the presence of the polymorphism makes to the mobility of thepolynucleotide or protein during gel electrophoresis. In the case of apolynucleotide single-stranded conformation polymorphism (SSCP) analysismay be used. This measures the mobility of the single strandedpolynucleotide on a denaturing gel compared to the correspondingwild-type polynucleotide, the detection of a difference in mobilityindicating the presence of the polymorphism. Denaturing gradient gelelectrophoresis (DGGE) is a similar system where the polynucleotide iselectrophoresed through a gel with a denaturing gradient, a differencein mobility compared to the corresponding wild-type polynucleotideindicating the presence of the polymorphism.

The presence of the polymorphism may be determined using a fluorescentdye and quenching agent-based PCR assay such as the Taqman PCR detectionsystem. In brief, this assay uses an allele specific primer comprisingthe sequence around, and including, the polymorphism. The specificprimer is labeled with a fluorescent dye at its 5′ end, a quenchingagent at its 3′ end and a 3′ phosphate group preventing the addition ofnucleotides to it. Normally the fluorescence of the dye is quenched bythe quenching agent present in the same primer. The allele specificprimer is used in conjunction with a second primer capable ofhybridizing to either allele 5′ of the polymorphism.

In the assay, when the allele comprising the polymorphism is present TaqDNA polymerase adds nucleotides to the nonspecific primer until itreaches the specific primer. It then releases polynucleotides, thefluorescent dye and quenching agent from the specific primer through itsendonuclease activity. The fluorescent dye is therefore no longer inproximity to the quenching agent and fluoresces. In the presence of theallele which does not comprise the polymorphism the mismatch between thespecific primer and template inhibits the endonuclease activity of Taqand the fluorescent dye in not released from the quenching agent.Therefore by measuring the fluorescence emitted the presence or absenceof the polymorphism can be determined.

In another method of detecting the polymorphism a polynucleotidecomprising the polymorphic region is sequenced across the region whichcontains the polymorphism to determine the presence of the polymorphism.

Accordingly, any of the following techniques may be utilized in thepresent methods for genotyping, as is known in the art.

-   -   General: DNA sequencing, sequencing by hybridization;    -   Scanning: PTT (Protein truncation technique), SSCP (single        strand conformational analysis), DGGE (denaturing gradient gel        electrophoresis), TGGE (temperature gradient gel        electrophoresis), Cleavase, Heteroduplex analysis, CMC (chemical        mismatch cleavage), enzymatic mismatch cleavage;    -   Hybridization based: solid phase hybridization (dot blots,        MASDA, reverse dot blots, oligonucleotide arrays (chips));        solution phase hybridization (Taqman, Molecular Beacons);    -   Extension based: ARMS (Amplification Refractory Mutation        System), ALEX (Amplification Refractory Mutation System Linear        Extension) SBCE (Single Base Chain Extension)    -   Incorporation based: Mini-sequencing, APEX; (Arrayed Primer        Extension)    -   Restriction enzyme based: RFLP (restriction fragment length        polymorphism)    -   Ligation based: OLA (Oligonucleotide Extension Assay)    -   Other: Invader (Third Wave Technologies).

The present invention also provides for a predictive (patient care) testor test kit. This predictive test could be a product and/or a servicewhich aids in disease management of asthma based on pre-determinedassociations between genotype and phenotypic response to leukotrienereceptor antagonists in treating asthma. Such a test could take twodifferent formats:

A) a molecular test which analyses DNA or RNA for the presence ofpre-determined polymorphisms. An appropriate test kit may include one ormore of the following reagents or instruments: a means to detect thebinding of the agent to the polymorphism, an enzyme able to act on apolynucleotide (typically a polymerase or restriction enzyme), suitablebuffers for enzyme reagents, PCR primers which bind to regions flankingthe polymorphism, a positive or negative control (or both), a gelelectrophoresis apparatus and a means to isolate DNA from a sample. Theproduct may utilise one of the chip technologies as described by thecurrent state of the art. The test kit would include printed or machinereadable instructions setting forth the correlation between the presenceof a specific polymorphism or genotype and the likelihood that a subjectwith asthma will respond favorably to therapy with a leukotrienereceptor antagonist; or

B) a biochemical test which analyses materials derived from thesubject's body, including proteins or metabolites, that indicate thepresence of a pre-determined polymorphism. An appropriate test kit wouldcomprise a molecule, aptamer, peptide or antibody (including an antibodyfragment) that specifically binds to a predetermined polymorphic region(or a specific region flanking the polymorphism), or a binding agent asdefined herein. The product may additionally comprise one or moreadditional reagents or instruments (as are known in the art). The testkit would also include printed or machine-readable instructions settingforth the correlation between the presence of a specific polymorphism orgenotype and the likelihood that a subject with asthma will respondfavorably to therapy with a leukotriene receptor antagonist.

The invention provides a method for screening a subject diagnosed withasthma potentially treatable by leukotriene receptor antagonists, todetermine the likelihood they will respond in a particular way totreatment with such a drug, more particularly a CysLT1 leukotrienereceptor antagonist and most particularly zafirlukast. The methodcomprises screening the subject for a polymorphism in the ALOX5 geneand/or the LTC4S gene that has previously been associated with a high orlow incidence of a particular desirable therapeutic outcome (compared tothe incidence in subjects with other genotypes). Subjects are mammalian,and preferably humans.

Treatment of a subject with a leukotriene receptor antagonist comprisesadministration of an effective amount of the pharmaceutical agent to asubject in need thereof. The dose of agent is determined according tomethods known and accepted in the pharmaceutical arts, and can bedetermined by those skilled in the art. A suitable dosage range forzafirlukast are provided in the disclosure of the Physician's DeskReference, the entire disclosure of which is hereby incorporated hereinby reference.

Genetic testing (also called genetic screening or genotyping) can bedefined broadly as analyzing the nucleic acid of a subject to determineif the subject carries mutations (or alleles or polymorphisms) that areeither (a) associated with, or causative of, a particular clinicalphenotype, or (b) that are in ‘linkage disequilibrium’ with a mutation,allele or polymorphism that is associated with or causative of aparticular clinical phenotype. One such clinical phenotype is thelikelihood that the subject will respond favorably to a giventherapeutic treatment.

Linkage disequilibrium refers to the tendency of specific alleles tooccur together more frequently than would be expected by chance. Allelesat given loci are in equilibrium if the frequency of any particular setof alleles (or haplotype) is the product of their individual populationfrequencies. Disequilibrium may be due to various forces, includingselection for certain allele combinations, or a recent mixing ofgenetically heterogeneous populations. Where markers link tightley to adisease-causing gene, an association of an allele (or a group of linkedalleles) with the disease gene is expected if the disease mutationoccurred in the recent historical past, so that sufficient time has notelapsed for equilibrium to be achieved through recombination events inthe immediate chromosomal region.

The term ‘marker,’ as used herein, refers to a specific site in thegenome which exhibits sequence variations among individuals.

The term ‘allele’ refers to the different sequence variants found atgiven markers. The sequence variants may be single or multiple basechanges, including insertions, deletions or substitutions or may bevariable number of sequence repeats and the like.

The term ‘linkage disequilibrium’ refers to the co-inheritance of twoalleles at frequencies greater than would be expected from the separatefrequencies of occurrence of each allele in a given control population.The expected frequency of occurrence of two alleles that are inheritedindependently is the frequency of the first allele multiplied by thefrequency of the second allele. Alleles that co-occur at expectedfrequencies are said to be in ‘linkage equilibrium.’ It will be apparentto those skilled in the art that the present methods may be carried outwith polymorphisms that are in linkage disequilibrium with the specificpolymorphisms identified herein.

The term ‘haplotype’ is a set of alleles that are inherited together asa group (i.e., that are in linkage disequilibrium). It will be apparentto those skilled in the art that the present methods may be utilized asa component of testing for haplotypes that encompass the polymorphismsdescribed herein.

EXAMPLES Example 1 Effects of Polymorphisms in the Promoter Region of5-Lipoxygenase and LTC4 Synthase on the Clinical Response (PhenotypicResponse) to Zafirlukast and Fluticasone

Asthma subjects were genotyped by two distinct polymorphisms in thepromoter regions of 5-lipoxygenase (ALOX5) and LTC4 synthase (LTC4S)genes. The polymorphisms were: the number of Sp1 repeats (n=3, 4, 5 or6) in the approximate region of 176 to 147 base pairs upstream from theATG start site in ALOX5 (Sp1), and an adenine to cytosine transversion444 nucleotides upstream from the first codon in LTC4S (A-444C). GenomicDNA was isolated from blood samples obtained from consenting subjectsparticipating in a 12 week multicenter, randomized, double-blind,double-dummy, parallel study comparing inhaled fluticasone (88 mcg BID)and oral zafirlukast (20 mg BID). Genomic DNA was extracted usingstandard procedures (automated extraction or using kit formats). Thegenotypes of the subjects, and any control individuals utilized, weredetermined for polymorphisms within the ALOX5 or LTC4S gene sequences.Polymorphisms are identifiable using PCR, PCR-RFLP, Taqman allelicdiscrimination assays, or any other suitable technique as is known inthe art. If a specific polymorphism resides in an amplification productthat is of sufficient physical size (e.g., an insertion/deletionpolymorphism of multiple bases), a simple size discrimination assay canbe employed to determine the genotype of an individual. In this case,two primers are employed to specifically amplify the gene of interest ina region surrounding the site of the polymorphism. PCR amplification iscarried out, generating products which differ in length, dependent onthe genotype (insertion or deletion) they possess. When subjected to gelelectrophoresis, the differently sized products are separated,visualized, and the specific genotypes interpreted directly.

PCR-RFLP (polymerase chain reaction—restriction fragment lengthpolymorphism) assays may also be utilized as is known in the art todetect polymorphisms. For each polymorphic site, a PCR-RFLP assayemploys two gene-specific primers to anneal to, and specifically amplifya segment of genomic DNA surrounding the polymorphic site of interest.Following PCR amplification, specific restriction endonuclease enzymesare employed to digest the PCR products produced. The enzyme utilizedfor an assay is selected due to its specific recognition sequence whichit requires to bind to, and cleave the PCR product in thepresence/absence of the polymorphism, yielding fragments diagnostic ofthe specific base present at the polymorphic site. Following cleavage bythe restriction enzyme, gel electrophoresis is employed to separate andvisualize the fragments produced.

Taqman assays, as are known in the art, may also be utilized to identifypolymorphisms. For each polymorphic site the allelic discriminationassay uses two allele specific probes labeled with a differentfluorescent dye at their 5′ ends but with a common quenching agent attheir 3′ ends. Both probes have a 3′ phosphate group so that Taqpolymerase cannot add nucleotides to them. The allele specific probescomprising the sequence encompassing the polymorphic site and willdiffer only in the sequence at this site (this is not necessarily true,the allele-specific probes can be shifted relative to each other suchthat they are not identical in length or composition. However, wherethey cover the same DNA region they are identical apart from thepolymorphic site of interest). The allele specific probes are onlycapable of hybridizing without mismatches to the appropriate site.

The allele specific probes are used in conjunction with two primers, oneof which hybridizes to the template 5′ of the two specific probes,whilst the other hybridizes to the template 3′ of the two probes. If theallele corresponding to one of the specific probes is present, thespecific probe will hybridize perfectly to the template. The Taqpolymerase, extending the 5′ primer, will then remove the nucleotidesfrom the specific probe, releasing both the fluorescent dye and thequenching agent. This will result in an increase in the fluorescencefrom the dye no longer in close proximity to the quenching agent.

If the allele specific probe hybridizes to the other allele the mismatchat the polymorphic site will inhibit the 5′ to 3′ endonuclease activityof Taq and hence prevent release of the fluorescent dye.

The AB17700 sequence detection system is used to measure the increase inthe fluorescence from each specific dye at the end of the thermalcycling PCR directly in PCR reaction tubes. The information from thereactions is then analyzed. If an individual is homozygous for aparticular allele only fluorescence corresponding to the dye from thatspecific probe will be released, but if the individual is heterozygous,then both dyes will fluoresce.

Primers and probes used in the present studies were as follows:

(SEQ ID NO:6) ALOX5 forward primer: AGGAACAGAC ACCTCGCTGA GGAGAG (SEQ IDNO:7) ALOX5 reverse primer: GAGCAGCGAG CGCCGGGAGC CTCGGC (SEQ ID NO:8)LTC4S-444A probe: CCTGGATGGG GACAGGGAAC AG (SEQ ID NO:9) LTC4S-444Cprobe: TGGATGGGGA CCGGGAACAG (SEQ ID NO:10) LTC4S forward primer:TCCGCAGAGG AGGGTTTG (SEQ ID NO:11) LTC4S reverse primer: GCTAACTCCTCCACCCACC T T

All patients met the ATS American Thoracic Society criteria for asthma,had a baseline forced expiratory volume (FEV₁) between 50 and 80%predicted, increased FEV1 by ≧12% following 180 mcg of inhaledalbuterol, and had not used inhaled or oral steroids within 6 months ofscreening. The endpoint predose a.m. FEV₁ results (given in the tablebelow as a % predicted change from baseline) by genotype and bytreatment are presented below in Table 1 (see also FIG. 2 for ALOX5;FIG. 3 for LTC4S):

TABLE 1 ALOX5 Sp1 LTC4S A-444C 5,5 5,X X,X A/A A/C C/C Fluticasone 14.2± 2.7 13.8 ± 2.0 8.3 ± 6.9 13.3 ± 2.3 14.8 ± 2.8 15.3 ± 6.2 n = 44 n =31 n = 5 n = 49 n = 20 n = 7 Zafirlukast 5.9 ± 1.8* 7.1 ± 3.8 −2.0 ± 3.25.3 ± 2.3* 5.9 ± 2.3* −1.8 ± 3.6 n = 44 n = 19 n = 5 n = 34 n = 30 n = 4*= Significantly different from fluticasone Where: A is the wildtypepolymorphic form of the LTC4S gene promoter region. C is the variantpolymorphic form of the LTC4S gene promoter region described in theSpecification above. 5 is the wildtype polymorphic form of the ALOX5gene promoter region. X is the variant polymorphic form of the ALOX5gene promoter region described in the specification above. n is thenumber of test subjects.

None of the subjects who were homozygous variant at ALOX5 (X/X) werealso homozygous variant for LTC4 (C/C). Some subjects were homozygousvariant for one gene and heterozygous variant for the other, and otherswere heterozygous variant for both genes.

The results indicate that subjects homozygous for variants in thepromoter region of either ALOX5 (X,X), or LTC4X (C/C), have a greatlyreduced response to the leukotriene receptor antagonist zafirlukastcompared to the other genotypes. These genes encode enzymes active inthe biosynthesis of sulfidopeptide leukotrienes. The response tofluticasone 88 mcg BID was greater than to zafirlukast 20 mg BID acrossall genotypes. These results indicate a genetic basis for some of thevariability observed in the clinical efficacy of the leukotrienereceptor antagonist zafirlukast.

For ALOX5, it was found that 59.5% of the patients were homozygous wildtype, 33.8% were heterozygous, and 6.8% were homozygous variant. Thesevalues are consistent with Drazen et al. who found frequencies of 56.1%,35.1% and 8.8% for ALOS5 homozygous wild type, heterozygous andhomozygous variant respectively. The frequencies for the LTC4S gene were50.0%, 44.1% and 5.90% for wild type homozygous, heterozygous andhomozygous variant, respectively. These values are similar to reportedvalues of 43.2, 50.5 and 6.3%, respectively (Sanar, M. et al. The Lancet1997; 350: 1599-1600).

Example 2 ALOX5 Polymorphisms at Nucleotide Positions 1728 and 1708Response to Zafirlukast and Fluticasone

The A to G transversion polymorphism at position 1728 in exon 13 of theALOX5 gene, and the G to A transversion polymorphism at position-1708,were studied for any association with the FEV1 response to zafirlukast,compared to the inhaled glucocorticoid fluticasone. Both polymorphismsare set forth in In, KH et al., J. Clin. Investigation 99 (5):1130(1997), the entire disclosure of which is incorporated herein byreference.

Genotyping was carried out on subjects with asthma participating in a 12week randomized, double blind, parallel study of fluticasone (88 mcgBID) and the leukotriene receptor antagonist zafirlukast (20 mg BID).Predose FEV1 results (change from baseline in percent predicted) areshown in Table 3.

TABLE 3 Fluticasone Zafirlukast Genotype G1708A G/G 15.0 ± 2.3 4.7 ±1.9* n = 50 n = 46 G/A 12.7 ± 2.3 8.5 ± 3.5 n = 26 n = 18 A/A — 0.7 ±2.2 n = 0 n = 4 Genotype A1728G A/A 13.6 ± 1.8 6.8 ± 1.7 n = 72 n = 58A/G 10.8 ± 6.3 −1.9 ± 2.9 n = 8 G/G — 0.8 n = 0 n = 1 *= significantdifference between zafirlukast and fluticasone

The above results suggest that subjects homozygous for the A allele atthe 1708 site (G1708A), or who have one or two G alleles at the 1728site (A1728G), had reduced response to zafirlukast, compared toalternative genotypes. This result did not reach statisticalsignificance, possibly due to low sample number.

The G1708A promoter polymorphism was in significant LinkageDisequilibrium (LD) with the Sp1 promoter marker that alters efficacy.The A1728G polymorphism was not in LD with the Sp1 site and thereforemay be affecting clinical response independently. The frequency of the Gand A alleles for 1708 are 0.82 and 0.18 respectively, and the A and Galleles for 1728 are 0.93 and 0.07 respectively. Both are in HardyWeinberg equilibrium. These results suggest that variability in clinicalresponse to zafirlukast may be associated with multiple geneticpolymorphisms in the leukotriene pathway.

1. A method of screening a subject suffering from asthma, as an aid in predicting their response to treatment with a leukotriene receptor antagonist ligand, comprising: a) obtaining a sample of DNA from the subject; and b) genotyping said DNA sample in the 5′ non-coding region of the LTC₄ Synthase (LTC4S) gene for the presence of SEQ ID NO:3 or SEQ ID NO:4; wherein homozygosity for an allele comprising SEQ ID NO:4 indicates that the subject is less likely to respond favorably to treatment with a leukotriene receptor antagonist for asthma, compared to a subject having an allele comprising SEQ ID NO:3.
 2. A method according to claim 1 where said leukotriene receptor antagonist ligand is a cysteinyl leukotriene 1 (CysLT1) receptor antagonist.
 3. A method according to claim 1 where said leukotriene receptor antagonist ligand is selected from the group consisting of zafirlukast, pranlukast, iralukast and montelukast.
 4. A method of identifying, within a population of asthma patients, a subpopulation of asthma patients with an increased likelihood of responding favorably to therapy with a leukotriene receptor antagonist, comprising the step of: (a) obtaining a DNA sample from each of said subjects; and; (b) conducting on each DNA sample a genotyping test selected from (i) determining the number of repeats of SEQ ID NO:12 in the 5′ noncoding region of the ALOX5 gene, and (ii) determining whether the 5′ non-coding region of the LTC₄ Synthase (LTC4S) gene is homozygous for an allele comprising SEQ ID NO:4 or contains an allele comprising SEQ ID NO:3; wherein the subpopulation of asthma patients with an increased likelihood of responding favorably to treatment with a leukotriene receptor antagonist consists of subjects with an ALOX5 allele having five repeats of SEQ ID NO:12 or having at least one LTC4S allele comprising SEQ ID NO:3.
 5. A method according to claim 4 where said leukotriene receptor antagonist ligand is a cysteinyl leukotriene 1 (CysLT1) receptor antagonist.
 6. A method according to claim 4 where said leukotriene receptor antagonist ligand is selected from the group consisting of zafirlukast, pranlukast, iralukast and montelukast. 